Abstract
Here we describe a protocol for the detection of the microRNA (miRNA) expression profile of a single cell by stem-looped real-time PCR, which is specific to mature miRNAs. A single cell is first lysed by heat treatment without further purification. Then, 220 known miRNAs are reverse transcribed into corresponding cDNAs by stem-looped primers. This is followed by an initial PCR step to amplify the cDNAs and generate enough material to permit separate multiplex detection. The diluted initial PCR product is used as a template to check individual miRNA expression by real-time PCR. This sensitive technique permits miRNA expression profiling from a single cell, and allows analysis of a few cells from early embryos as well as individual cells (such as stem cells). It can also be used when only nanogram amounts of rare samples are available. The protocol can be completed in 7 d.
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References
Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004).
Harfe, B.D. MicroRNAs in vertebrate development. Curr. Opin. Genet. Dev. 15, 410–415 (2005).
Alvarez-Garcia, I. & Miska, E.A. MicroRNA functions in animal development and human disease. Development 132, 4653–4662 (2005).
Plasterk, R.H. Micro RNAs in animal development. Cell 124, 877–881 (2006).
Krichevsky, A.M., King, K.S., Donahue, C.P., Khrapko, K. & Kosik, K.S. A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 9, 1274–1281 (2003).
Nelson, P.T. et al. Microarray-based, high-throughput gene expression profiling of microRNAs. Nat. Methods 1, 155–161 (2004).
Thomson, J.M., Parker, J., Perou, C.M. & Hammond, S.M. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1, 47–53 (2004).
Baskerville, S. & Bartel, D.P. Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 11, 241–247 (2005).
Liang, R.Q. et al. An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe. Nucleic Acids Res. 33, e17 (2005).
Barad, O. et al. MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res. 14, 2486–2494 (2004).
Babak, T., Zhang, W., Morris, Q., Blencowe, B.J. & Hughes, T.R. Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 10, 1813–1819 (2004).
Miska, E.A. et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol. 5, R68 (2004).
Calin, G.A. et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11755–11760 (2004).
Sun, Y. et al. Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs. Nucleic Acids Res. 32, e188 (2004).
Sioud, M. & Rosok, O. Profiling microRNA expression using sensitive cDNA probes and filter arrays. Biotechniques 37, 574–576–578–580 (2004).
Liu, C.G. et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc. Natl. Acad. Sci. USA 101, 9740–9744 (2004).
Sempere, L.F. et al. Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol. 5, R13 (2004).
Lu, J. et al. MicroRNA expression profiles classify human cancers. Nature 435, 834–838 (2005).
Neely, L.A. et al. A single-molecule method for the quantitation of microRNA gene expression. Nat. Methods 3, 41–46 (2006).
Chen, C. et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 33, e179 (2005).
Tang, F., Hajkova, P., Barton, S.C., Lao, K. & Surani, M.A. MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res. 34, e9 (2006).
Lao, K. et al. Multiplexing RT-PCR for the detection of multiple miRNA species in small samples. Biochem. Biophys. Res. Commun. 343, 85–89 (2006).
Handyside, A.H., Kontogianni, E.H., Hardy, K. & Winston, R.M. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 344, 768–770 (1990).
Zamore, P.D. & Haley, B. Ribo-gnome: the big world of small RNAs. Science 309, 1519–1524 (2005).
Vazquez, F. et al. Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs. Mol. Cell 16, 69–79 (2004).
Yi, R. et al. Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat. Genet. 38, 356–362 (2006).
Kanellopoulou, C. et al. Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing. Genes Dev. 19, 489–501 (2005).
Murchison, E.P., Partridge, J.F., Tam, O.H., Cheloufi, S. & Hannon, G.J. Characterization of Dicer-deficient murine embryonic stem cells. Proc. Natl. Acad. Sci. USA 102, 12135–12140 (2005).
Cobb, B.S. et al. T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer. J. Exp. Med. 201, 1367–1373 (2005).
Muljo, S.A. et al. Aberrant T cell differentiation in the absence of Dicer. J. Exp. Med. 202, 261–269 (2005).
Acknowledgements
We are grateful to N.L. Xu, C. Chen, V. Yeung, N. Straus, K. Livak and N. Miyoshi for their helpful suggestions. The work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences Research Council (BBSRC) to M.A.S.
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Tang, F., Hajkova, P., Barton, S. et al. 220-plex microRNA expression profile of a single cell. Nat Protoc 1, 1154–1159 (2006). https://doi.org/10.1038/nprot.2006.161
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DOI: https://doi.org/10.1038/nprot.2006.161
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